Tumor necrosis factor (TNF) is a pleiotropic cytokine involved in the regulation of numerous physiological and pathological processes such as inflammation, cancer, autoimmunity and infection. TNF is also linked to the development of various neurological disorders, including multiple sclerosis (MS). TNF exists in two biologically active forms, transmembrane (tmTNF) and soluble (solTNF), whose functions are mediated by TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). By virtue of their different binding affinities, solTNF is the primary signaling partner of TNFR1, while tmTNF preferentially signals through TNFR2. The cellular processes activated by the two receptors are often opposite: TNFR1 mediates apoptosis and chronic inflammation, TNFR2 mediates cell survival, resolution of inflammation, immunity and myelination. Numerous studies have underscored the importance of distinguishing between the functions of solTNF and tmTNF, and have associated MS and its animal model experimental autoimmune encephalomyelitis (EAE) to the detrimental effects of solTNF and TNFR1. In our own studies we have demonstrated not only that solTNF is detrimental, but that tmTNF is protective and important for repair and remyelination in EAE. We showed that mice treated with the biologic XPro1595, a dominant negative TNF that selectively blocks solTNF without affecting tmTNF, recover from EAE-induced paralysis. This is associated with neuroprotection, improved myelin integrity and increased remyelination. How does this occur? Our HYPOTHESIS is that selective inhibition of solTNF causes a shift in the balance of TNF receptor activation towards TNFR2. This leads to the downstream activation of TNFR2-dependent cascades which carry out the protective functions of tmTNF in EAE. Since in the CNS TNFR2 is expressed in oligodendrocytes, oligodendrocyte precursors, astrocytes and microglia, all these cell populations could potentially be the effectors of tmTNF beneficial effect. In this proposal we will focus exclusively on microglia. We have found that TNFR2 is expressed in microglia, both in mouse EAE and in MS. Activation of TNFR2 in microglia has been shown to induce the production of anti-inflammatory and neuroprotective mediators. On this basis, we hypothesize that activation of TNFR2 in microglia by tmTNF sets off protective signaling cascades that contribute to tmTNF beneficial effects. The OBJECTIVE of our studies is to determine whether microglial TNFR2 is a mediator of tmTNF protective function in EAE. We will address this question with an innovative genetic approach consisting of a new mouse model generated in our laboratory where TNFR2 is ablated from the myeloid population (LysMcreTNFR2fl/fl mice) to obtain bone-marrow chimeras that will allow for specific investigation of microglial TNFR2. These experiments will shed light on tmTNF-TNFR2 signaling in microglia in vivo, a question that has not been addressed to date. We believe our studies will significantly advance the current knowledge on the role of tmTNF and TNFR2 in EAE, providing the basis for the possible development of a selective solTNF inhibitor as a new therapy for MS.